Transgenic zebra fish embryo model for hematopoiesis and lymphoproliferative disorders

ABSTRACT

A transgenic zebrafish animal model for the study of haemopoietic cell differentiation, control, and screening of therapeutic agents.

BACKGROUND OF THE INVENTION

Acute lymphoblastic leukemia (ALL) is the most common form of cancer inchildren (Pizzo and Poplack, 1993, Greaves, 1986, Uckun et al., 1998,Crist et al., 1988). A better understanding of the biological basis andpredisposing leukemogenic events in this disease is needed in order todevelop more effective treatment programs as well as novel preventionstrategies.

Leukemic clones are thought to originate in ALL patients from normallymphocyte precursors arrested at various stages of T- or B-lymphocytedevelopment (Greaves, 1986). Accordingly, any critical regulatorynetwork that controls normal lymphocyte development is a potentialtarget for a leukemogenic event.

One such regulatory network vital for normal hematopoiesis involvesIkaros, a member of the Kruppel family “zinc finger” DNA-bindingproteins. Ikaros acts as an evolutionarily conserved “master switch” ofhematopoiesis that dictates the transcriptional regulation of lymphocyteontogeny and differentiation (Georgopoulos et al., 1994, Georgopoulos etal., 1992, Hahm et al., 1994, Molnar and Georgopoulos, 1994, Wang etal., 1996, Winandy et al., 1995, Molnar et al., 1996, Sun et al., 1996,Hansen et al., 1997, Georgopoulos et al., 1997, Brown et al., 1997, Kluget al., 1998).

The programmed expression and function of the Ikaros gene is tightlycontrolled by alternative splicing of the Ikaros pre-mRNA which resultsin production of eight different Ikaros isoforms. All eight Ikarosisoforms share a common carboxy(C)-terminal domain containing 1transcription activation motif and two zinc finger motifs that arerequired for hetero- and homodimerization among the Ikaros isoforms andfor interactions with other proteins (Hahm et al., 1994, Molnar andGeorgopoulos, 1994, Sun et al., 1996). Only three of the eight Ikarosisoforms, however, contain the requisite three or more amino(N)-terminalzinc fingers that confer high affinity binding to an Ikaros-specificcore DNA sequence motif in the promoters of target genes (Sun et al.,1996).

The formation of homo- and heterodimers among the DNA binding isoformsincreases their affinity for DNA, whereas heterodimers between the DNAbinding isoforms and non-DNA binding isoforms are unable to bind DNA.Therefore, Ikaros proteins with fewer than three N-terminal zinc fingersexert a dominant negative effect by interfering with the activity ofIkaros isoforms that can bind DNA (Molnar et al., 1996, Sun et al.,1996). Thus, splicing errors can have severe consequences for thelymphocyte compartment of the developing immune system. An abundance ofdominant-negative Ikaros isoforms that no longer bind DNA could resultin significantly impaired expression of regulatory target genes that areessential for the orderly development and maturation of lymphocyteprecursors.

In mice, absence of the normal Ikaros gene results in an early andcomplete arrest in the development of all lymphoid lineages during bothfetal and adult hematopoiesis (Georgopoulos et al., 1994).Ikaros-deficient mice have a rudimentary thymus, lack peripheral lymphnodes, and are characterized by a complete absence of lymphocyteprogenitor cells as well as mature B-lymphocytes, T-lymphocytes, andnatural killer cells (Georgopoulos et al., 1994). Mice heterozygous fora germline mutation which results in the loss of critical DNA-bindingzinc fingers of Ikaros develop a very aggressive form of lymphoblasticleukemia with a concomitant loss of the single wild type Ikaros allelebetween three and six months after birth (Winandy et al., 1995).Finally, the most recent findings in ALL molecular etiology show apivotal role for Ikaros gene regulation in lymphoblast neoplastictransformation in infants (Sun et al., 1999) with T-lineage or B-lineageALL leukemic cells expressing high levels of dominant-negative Ikarosisoforms.

It has long been suspected that molecular rearrangements in the lymphoidlineage precursors leading to ALL occur during fetal hematopoiesis (Fordet al., 1993, Gill Super et al., 1994). With the prospect of Ikarosmalfunction and Ikaros isoform expression being at the core ofleukemogenesis, a better understanding of the events taking place duringembryonic blood cell differentiation is required in order to developrational therapies. To address this need, an adequate experimental modelsystem of vertebrate hematopoiesis is essential.

The zebrafish (ZF), with its extremely rapid embryonic development (3days) and short maturation period (2-3 months) offers an attractivemodel. Over the past decade, the ZF embryo has been used to studyeukaryotic gene activity and intercellular signaling in vertebratedevelopment (Nusslein-Volhard, 1994, Zhang et al., 1998, Nguyen et al.,1998), and has emerged as a powerful genetic system, strongly relevantto the study of molecular medicine (Driever and Fishman, 1996, Amemiya,1998). Intensive study of early embryonic hematopoiesis in the ZF alongwith the generation of hematopoietic mutants has turned the ZF into auseful model for the study of human blood disorders, such as congenitalsideroblastic anemia (Brownlie et al., 1998) and hepatoerythropoieticporphyria (Wang et al., 1998). (See detailed reviews: Bahary and Zon,1998, Amatruda and Zon, 1999).

It has now been discovered that transient, inappropriate expressionduring early embryonic development of the non-DNA binding Ikaros forms,including the dominant-negative isoforms, mutant forms, and others, havea significant impact on blood cell differentiation at later stages ofdevelopment. Using the transgenic animal model of the invention, theeffect of various agents on blood cell differentiation can beefficiently assessed. The ZF, with its relatively large and translucentembryo, external fertilization, and extracorporate development, providesa model of choice for transgenic research (Stuart et al., 1990, Culp etal., 1991, Hammerschmidt et al., 1999).

This model can be used, for example, to examine the impact of alterationof the Ikaros program of gene expression on definitive hematopoiesis inadults, within the short period of hematopoietic cell determination inZF embryonic development.

As described herein, a transgenic Zebrafish (ZF) animal model providesan excellent model of vertebrate hematopoiesis.

SUMMARY OF THE INVENTION

The present invention provides a useful animal model for the screeningand study of hematopoiesis and agents capable of modulatinghematopoietic development. In particular, the ZF embryo carrying anIkaros transgene provides a model for the study and modulation oflymphocyte development and leukemia.

In one embodiment of the invention, the transgene is a DNA-bindingIkaros isoform, for example, Ik-2. The ZF embryo animal model carryingthe Ik-2 transgene can be used to screen and identify agents thatinterfere with or overcome normal Ik-2 function, for example, inducingB- or T-cell cancers, particularly leukemia. Potential cancer-inducingagents such as proteins, gene alterations, pharmaceuticals, toxins, andthe like, are screened by administration to the model, and thedisruption of normal function is monitored. An Ik-2 transgenic ZF embryomodel thus can provide a screening assay for potential carcinogens.

In an alternative embodiment of the invention, the ZF embryo istransformed with a non-DNA binding form of Ikaros. The non-DNA bindingform can be, for example, Ik-4, 5, 6, 7, 8, 9, or 10, each of whichlacks the three N-terminal zinc fingers required to confer high affinityDNA binding. A mutant Ikaros protein can also be used, for example thoseIk deletion and insertion mutants described in PCT Patent ApplicationPCT/US99/26274 and discussed more fully below. Because the ZF modelcontaining a non-DNA binding Ik, e.g. Ik-4, develops leukemia at laterstages of development, it can be used to screen for preventative andtherapeutic agents.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A1-1D3 are computer generated images demonstrating successfulmicroinjection and expression of human Ikaros proteins in Zebrafishembryos.

FIGS. 1A2, and 1A3 show a gfp-positive ZF embryo at mid gastrula stage,6 hpf (FIG. 1A 1), and Ikaros cDNA (FIG. 1A 2) and control beta actincDNA (FIG. 1A 3) amplified from embryonic mRNA.

FIGS. 1B1-1B4 show a gfp-positive ZF embryo at prim-5 stage, 24 hpf(FIG. 1B 1), and Ikaros cDNA (FIG. 1B 2) and control beta actin cDNA(FIG. 1B 3, 1B4) amplified from embryonic mRNA.

FIGS. 1C1-1C3 show a gfp-positive ZF embryo at prim-5 stage, 24 hpf(FIG. 1C 1), and Ikaros cDNA (FIG. 1C 2) and control beta actin cDNA(FIG. 1C 3) amplified from embryonic mRNA.

FIGS. 1D1-1D3 show a gfp-positive ZF embryo at long-pec stage, 48 hpf(FIG. 1D 1), and Ikaros cDNA (FIG. 1D 2) and control beta actin cDNA(FIG. 1D 3) amplified from embryonic mRNA.

FIGS. 2A-2F are computerized photographic images demonstrating Ik-4 andIk-2 transgene expression in ZF embryos by whole-mount in situhybridization.

FIG. 2A shows a control embryo at 17 hpf with a negative hybridizationresult using the Ik-4 rhyboprobe.

FIGS. 2B-2D show Ik-4 injected embryos at 17 hpf, each demonstratingpositive hybridization result with Ik-4 expression in the trunk regionof the embryo. FIGS. 2E-2F are Ik-2 injected embryos at approximately 17hpf, demonstrating positive hybridization with Ik-2 expression in thetrunk region of the embryo.

FIG. 3A is a computerized photograph of a medium sagattale section ofthe trunk region of a 48 hpf ZF embryo showing anterior to the left anddorsal to the top of the frame and providing a layout of the “dorsal”hematopoietic site.

FIGS. 3B-3E show frozen sagattal sections of the embryo immuno stainedwith antibodies raised against human Ikaros.

FIG. 3B demonstrates Ik-4 immuno staining localized to the nuclei andcytoplasm in circulating blood cells, while FIG. 3C demonstrates Ik-4immuno staining in the mesenchymal hematopoietic cells of the dorsalaorta ventrical wall.

FIG. 3D demonstrates Ik-2 immuno staining localized to the nuclei incytoplasm of circulating blood cells. Specificity of the anti-Ikarosantibodies to the human protein was confirmed by control staining. Innon-injected ZF embryos, no cross reactivity with the andogenis ZFIkaros was observed (FIG. 3E).

FIGS. 4A-4O are computerized photographic images showing expressionpatterns of the ZF early hematopoietic genes GATA-1, c-MYB, Rag-1, andlck.

FIG. 4A is a control embryo at 17 hpf with GATA-1 positive cells in thetrunk region stained pink with fast red. No Ikaros staining is present.

FIG. 4B is a lateral view of an Ik-4 transgenic embryo at 17 hpf showingblue-purple spots in patches marking Ik-4 expression and a strong pinkstaining of the ICM region.

FIG. 4C is a lateral view of an Ik-2 transgenic embryo at 17 hpf showingblue-purple signals of the Ik-2 and no traces of GATA-1 expression.

FIG. 4D is a laser confocal image of the embryo shown in FIG. 4A, withfluorescent GATA-1 positive ICM staining.

FIG. 4E is a laser confocal image of the embryo shown in FIG. 4B, havinga dramatic increase in the ICM due to abnormal expansion of GATA-1positive cells.

FIG. 4F is a laser confocal image of the embryo shown in FIG. 4C,demonstrating a decline in GATA-1 expression.

FIG. 4G is a laser confocal image of the control embryo at 15 hpfshowing fluorescent c-MYB positive strip.

FIG. 4H is a laser confocal image of the Ik-4 transgenic embryo at 15hpf showing an expansion of the c-MYB positive cells in the cotal partof the ICM.

FIG. 4I is a laser confocal image of the Ik-2 transgenic at 15 hpf withnearly total failure of c-MYB expression.

FIG. 4J is a lateral view of the control 4 dpf ZF larvae with Rag-1positive cells localized to the area of pharangyl arches between the eyeand pectorial fin.

FIG. 4K is a lateral view of the Ik-4 injected larvae at 4 dpf withRag-1 positive thymic site.

FIG. 4L is a lateral view of the Ik-2 injected larvae at 4 dpf withRag-1 positive thymic site.

FIG. 4M is a control non-injected ZF larvae at 4 dpf with lck positivethymic site.

FIG. 4N shows an Ik-4 injected ZF larvae at 4 dpf with lck positivethymic site.

FIG. 4O shows an Ik-2 injected ZF larvae at 5 dpf without lck positivethymic site.

FIG. 5A is a graphic represention of hematopoietic cell indexescalculated for adult ZF derived from Ik-4 injected embryos (n=18), adultZF derived from Ik-2 injected embryos (n=22), and adult ZF derived fromcontrol non-injected embryos (n=10).

FIGS. 5B-5C are computerized microscopic images of kidney hematopoieticcells imprinted onto slides from intact embryos and differentiallystained with Wright/Giesma.

FIG. 5B shows imprinted cells of ZF kidney derived from Ik-2 injectedembryos.

FIG. 5D shows kidney hematopoietic cells from ZF derived from Ik-4injected embyos.

In FIGS. 5B, 5C, and 5D myeloid cells are marked as M, erythroblastcells are marked as E, and lymphoid cells are marked with arrowspointing to the cells.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention relates to the discovery that expression of Ikarosisoforms in ZF embryos provides modulation of hematopoietic celldevelopment. Such modification is specific, and correlates withdevelopment events in human hematopoietic cells and with specifichematopoietic disorders, including leukemias and anemias.

Accordingly, the transgenic ZF animal model described more fully in theexamples below provides a rapid and efficient screening assay for agentsthat modulate normal hematopoietic cell development. The animal model isthus useful for studying and understanding regulation of hematopoietic,as well as for screening agents for prevention and/or therapeuticapplications.

In a preferred method of the invention, a zebrafish embryo carrying atransgene encoding a DNA-binding or non-DNA binding Ikaros protein isprovided for analysis. ZF embryos expressing DNA binding Ikaros proteinare useful for studying normal B- and T-cell lineage development, andfor screening agents suspected of altering normal development. Inaddition, this model can be used to screen agents effective to increasered blood cell counts, as an indication of useful anti-anemia therapy.

ZF embryos expressing non-DNA binding isoforms (Ik-4, 5, 6, 7, 8 anddeletion or insertion lk mutants) can be used to screen for potentialtherapeutic candidates, particularly for the prevention and/or treatmentof hematopoietic disease. Examples of mutant Ikaros proteins includethose lacking the following amino acid sequence: KSSMPQKFLG [SEQ ID NO:5]. An exemplary insertion mutant is an Ikaros protein containing aninsertion of the following amino acid sequence: VTVGADDFRDFHAIIPKSFSR[SEQ ID NO 6]

EXAMPLES

The invention may be better understood with reference to the followingExamples. These are intended only to exemplify the invention, and not tolimit the scope of the invention in any way.

Example 1 Expression Constructs and Microinjection of Zebrafish Embryos

Fish and embryos. The adult wild type ZF were maintained generallyaccording to the Zebrafish book recommendations (Westerfield, 1995).Males and females were kept in 10 G tanks, 70 fish per tank, with aconstant slow flow of conditioned water at 26° C. and a controlled 14hours day/10 hours night cycle. Embryos were obtained through naturalspawning in breeding cages with a netted false bottom or by in vitrofertilization using eggs and milt collected from the mature females andmales anesthetized with tricaine (Sigma). Embryos were kept at 28.5° C.in Petry dishes, 30-50 per dish.

Five days after hatching, frys were transferred to a nursery for 2 weeksand raised in 1 G mouse cages at 28.5° C. Larvae were fed with livefood, paramecia and brine shrimps, according to recommendations of Dr.Stephen Ekker (personal communication). The survival rate was over 95%.Juvenile ZF were transferred to 10 G tanks, treated as adult fish asdescribed above and raised to maturity for another 3 months. For in situhybridization studies, embryos at 1-5 days post fertilization (dpf) weretreated with 0.003% phenylthiourea (Sigma) to prevent pigmentation.

Genes and expression vectors. Transgenic expression of human Ikarosisoforms Ik-2 and Ik-4 were driven by the promoter/enhancer region ofcarp β-actin gene in all-fish expression vector pFV4aCAT (Caldovic andHackett, 1995). The DNA vectors were constructed by cloning the humanwild type Ikaros cDNA Ik-2 and Ik-4 into pFV4aCAT at the NotI and SpeIsites. The vectors were re-named hIK4 wt pFV4aCAT and hIK2 wt pFV4aCAT,respectively. After further digestion with XbaI, the linearizedfragments containing Ikaros cDNA were then purified with a Spin X column(Costar) and used for microinjections.

Microinjections. Microinjections were performed with the help of aSMZ-10A stereo microscope (Nicon) and Transjector 5246 (Eppendorf) atroom temperature (RT) using glass micropipettes with splinted sharp tipsof 2-3 μm diameter. The eggs in chorions at the early one-cell stagewere positioned in grooves of agar-lined Petri dishes, as described inWesterfield, 1995.

The constructs were coinjected, according to the method recommended byHyatt and Ekkcer, 1999, with green fluorescent protein (GFP) mRNA (147.4ng/μL), to confirm the presence of the injected molecules and to enableselection of GFP-positive fish for future analyses. DNA was dissolved toa final concentration of 10 ng/μL in Hank's saline containing 0.01%phenol red, in order to follow the injection procedure. Approximately 5nl of the injected medium containing the equivalent of approximately 50pg (10⁷ copies) of the construct was injected into the cytoplasm of theegg's blastodisk under visual control. Each construct was injectedindependently in 3-5 series of injections and the data obtained waspooled. Following the injections, eggs were incubated at 28.5° C.

Embryo observations were carried out with a SMZ-10A stereo microscope(Nicon), equipped with an additional filter setting for fluorescencedetection and a specially designed transparent heating tray to keepembryos at constant temperature. Pictures of the embryos were taken witha H-III Photomicrographic System (Nicon) on Ektachrome 100× film(Kodak). Fluorescent embryos were imaged using MRC-1024 Laser ScanningConfocal Imaging System (Bio-Rad). The embryos and larvae were observedand analyzed at 6, 24, and 48 hours post fertilization (hpf) and at 3,4, and 5 days post fertilization (dpf), or were raised to adulthood forfuture analyses (see above).

mRNA Isolation and RT-PCR. mRNA was extracted from individual zebrafishembryos and adult fish tissues using the Oligotex™ Direct mRNA isolationKit (Qiagen, Valencia, Calif.). Possible DNA contamination waseliminated by incubating all mRNA with 1 μl DNase (Promega, RQ1 RNaseFree DNase) in 100 μl final volume of buffer containing 50 mM NaCl, 5 mMTris-Cl, pH7.5 at 37° C. for 30 minutes. The reactions were stopped byphenol/chloroform extraction and mRNA was precipitated with ethanol.Reverse transcription was done with a 500 ng mRNA template in the 20 μlfinal volume using Advantage™ RT-for-PCR Kit (Clontech). The cDNAobtained by reverse transcription was diluted to a total volume of 100μl by adding 80 μl H₂O. PCR amplification of Ikaros cDNA was achievedusing 10 μl of the diluted cDNA as template and the Advantage® cDNAPolymerase Mix (Clontech) in a 50 μl reaction volume. Primers foramplification of Ikaros cDNA are shown below: F1,5′-ATGGATGCTGACGAGGGTCAAGAC-3′ [SEQ ID NO: 1] and R1,5′-CTAGTGGAATGTGTGCTCCCCTCG-3′. [SEQ ID NO: 2]

The integrity of the mRNA and cDNA was confirmed by PCR amplification ofzebrafish β-actin cDNA in the same reaction with primers specific forβ-actin: ACTf, 5′-GATGATGCCCCTCGTGCTGTTTTC-3′ [SEQ ID NO: 3] and ACTr,5′-TTTCTCTTTCGGCTGTGGTGGTGA-3′. [SEQ ID NO: 4]

The 4 kb injected DNA fragment was used as a positive control PCRtemplate for comparison to the size of the amplified fragments.

Sectioning. Dechorionated or hatched ZF embryos at 48 hpf were fixed in4% paraformaldehyde/phosphate buffer (PBS) at 4° C. for 2 hours, soakedin 30% sucrose, embedded into OCT cryostat embedding medium (Fisher) andfrozen in liquid N₂ exactly as described previously in Westerfield,1995. Sections of 5 μm were prepared with the cryotome CM 1800 (Lieca).Slides with mounted ZF sections were stored at −80° C. until rehydratedby washing 3 times with PBS, pH 7.4 (Celox Laboratories). Sections werestained with hematoxyline and eosine as described in Luna, 1968, orsubjected to immunostaining.

Immunostaining. Tissue sections were permeabilized by soaking with ablocking solution of PBS containing 2.5% bovine serum albumin (BSA)(Sigma) and 0.1% Triton-X-100 (Fisher Biotech) for 30 minutes, and thentreated with primary antibodies (rabbit anti-Ikaros IgG (1:100), ParkerHughes Institute) for 1 hour at room temperature. Treated sections werewashed 3 times with PBS, stained with fluorescein isothiocyanate-labeledsecondary antibodies (donkey anti-rabbit Ig (1:40), Amersham) for 1 hourat room temperature and washed 3 times with PBS, after which Vectashieldmounting medium with propidium iodide (Vector Laboratories) andcoverslips were applied. Stained ZF embryo sections were imaged usingMRC-1024. Laser Scanning Confocal Imaging System (Bio-Rad).

Whole-mount in situ hybridizations were carried out on embryos at 15-19hpf and 4, 5 dpf according to the methods described in Jowett, 1999.Ikaros riboprobes were labeled with digoxigenin, whereas GATA-1, c-MYB,Rag-1, and lck probes, used in two-color/fluorescent in situhybridizations, were labeled with fluorescein. Synthesis of RNA probeswas performed by in vitro transcription using a DIG RNA Labeling Kit(Roche Molecular Biochemicals). For Ikaros riboprobes, human Ikaros cDNA(Ik-4 or Ik-2) used as a template was cloned to the pBluescript/KS+vector and linearized at the XhoI site. Anti-sense RNA was in vitrotranscribed with T7 RNA polymerase for 2 hours at 37° C. and labeled bydigoxigenin-11-UTP added to the nucleotide mixture. GATA-1 templatecloned to the pBlueskript/SK+ vector was linearized with XbaI. c-MYB orlck cloned to pBKCMV were linearized with EcoRI, and Rag 1 cloned to pCR2.1 vector was linearized with Hind III restriction endonuclease.

Anti-sense probes were synthesized with T7 RNA polymerase, as above.Sense probes used for control staining were synthesized with T3 RNApolymerase. Prior to in situ hybridization, the efficacy of reaction wasconfirmed by gel electrophoresis and Northern hybridization. Embryosfixed with 4% paraformaldehyde for 12 hours at 4° C. were graduallydehydrated in methanol and kept at −20° C. overnight. Followingrehydration, embryos were prehybridized for 5 hours at 70° C., thenhybridized with corresponding riboprobe(s) at 70° C. (overnight) andfinally treated with anti-DIG or anti-fluorescein Fab fragments forimmunolocalization of the haptens. Detection of the DIGantibody-alkaline phosphatase conjugate was carried out by staining withnitroblue tetrazoleum/5-bromo-4-chloro-3-indolilphosphate (NBT/BCIP)substrate mixture which produces an insoluble, blue-purple precipitate.Visualization of fluorescein antibodies conjugated to alkalinephosphatase complex was accomplished by staining with Fast Red(Boehringer Mannheim) which produces a precipitate that is bothchromogenic (pink) and fluorescent. After hybridization, embryos werere-fixed in 4% paraformaldehyde and photographed. Images of embryos werethen taken with an H-III Photomicrographic System (Nicon) on Ektachrome100× film (Kodak). Fluorescence in the embryos was detected and imagedusing MRC-1024 Laser Scanning Confocal Imaging System (Bio-Rad) mountedon a Nikon Eclipse E800 upright microscope with high numerical apertureobjectives. Digital data from 30-34 optical section series werecollected and 3D images were reconstructed using Lasersharp software(Bio-Rad, Hercules Calif.) and printed on a Fuji Pictrography thermaltransfer printer (Fuji, Elmsford, N.Y.).

Adult fish, tissues, and kidney imprints. The size, color, sex andweight of adult three-month-old fish were determined. Blood wascollected from the caudal vein from anesthetized fish and organs/tissues(spleens, intestines, brains, eyes, hearts, and kidneys) were dissectedand frozen in liquid Nitrogen for RT-PCR analysis. Prior to freezing,color and weight of spleens and livers were analyzed and weight indexeswere calculated (weight of the organ×100/total fish weight). Kidneyswere imprinted on slides which were air dried and stained withWright/Giemsa according to the University of Maryland Special HematologyLaboratory protocols. Kidney imprints were studied microscopically forcellular composition and photographed using a microscope Eclipse E800(Nicon) and a H-III Photomicrographic System (Nicon).

Cell morphology analysis. Blood cells were typed according to cellmorphology and PAS, Sudan black, and myeloperoxidase staining. Cellcounts in the kidney imprints were performed at ×100 magnification usingan eye-piece grid in 10 different areas of each sample. The number oflymphoid, myeloid, and erythroid cells as well as granulocytes andmonocytes was determined and cell indexes were calculated for each ofthe cell lineages.

Statistics. Data obtained from adult fish measurements and from kidneycell counts were checked for normal distribution and subjected to astandard Student's two-tailed test with Welch's correction, whennecessary. Statistical analysis and graphing were performed usingGraphPad Prism version 2.0 (GraphPad Software, Inc., San Diego, Calif.).

Example 2 Transgenic Expression of Human Ikaros Isoforms Ik-4 and Ik-2in ZF Embryos

Linearized expression vectors hIK4 wt pFV4aCAT and hIK2wt pFV4aCAT weremixed with GFP mRNA and microinjected into one-cell stage ZF embryos toforce expression of the dominant-negative human Ikaros isoform Ik-4 andthe DNA binding human Ikaros isoform Ik-2 (control) during primitivehematopoiesis. The microinjections were successful in >95% of allembryos, as evidenced by a strong green fluorescence documenting theexpression of the coinjected GFP mRNA from mid-gastrula until prim-5stage (FIGS. 1A1, 2A1, 3A1).

Total mRNA was extracted from GFP-positive individual ZF embryos at 6hpf, mid-gastrula stage (FIG. 1A 1), at 24 hpf, prim-5 stage (FIGS. 1B1,1C1) and at 48 hpf, Long-pec stage (FIG. 1D 1). The extracted mRNA wasreverse-transcribed using oligo-dT and random hexamers. The resultingcDNAs were amplified with ZF β-actin primers (FIGS. 1A3, 1B3, 1C3, and1D3) to test the integrity of the extracted mRNAs. Human Ikarosexpression was analyzed by amplification with Ikaros-specific primershILKEX7R1 and hIKEXIF (FIGS. 1A2, 1B2, 1C2, and 1D2).

RT-PCR analysis of total RNA from GFP-positive embryos (n=13-18 per timepoint) confirmed the time-dependent expression of human Ik-4 and Ik-2mRNA. At late blastula stage (4 hpf), no human Ikaros transgeneexpression had been detected, likely due to a lack of transcriptionalactivity of the zygotic genes (data not shown). In contrast, duringembryonic shield formation at mid-gastrula stage (6 hpf), prim-5 stage(24 hpf), and long-pec stage (48 hpf), 100% of the tested embryosexpressed the corresponding human Ikaros transgene (1A2, 1B2, and 1C2,respectivelly).

Expression of the human Ikaros transgenes Ik-4 and Ik-2 was transient.Only half of the hatching embryos (72 hpf), one third of the larvae (96hpf), and none of the adult ZF tissues showed RT-PCR evidence for humanIkaros transgene expression (data not shown). Microinjections did notsignificantly affect the viability and survival rate of the ZF embryos.Of 162 non-injected control embryos, 146 (90.1%) developed up tohatching without any visual abnormalities. Similarly, 316 of 361 (87.5%)GFP-positive embryos microinjected with the Ik-4 expression vector hIk4wtpFV4aCAT and 256 of 303 (84.5%) GFP-positive embryos microinjectedwith the Ik-2 expression vector hIk2 wtpFV4aCAT developed normally(Table 1). TABLE 1 Survial Rates and Embryonic Development followingIkaros injections # # Injected # GFP- # Normal # Normal OligochromenFertilized Positive Embryos Larval Larval Con- Eggs Embryos 3-48 hpf*72-96 hpf 72-96 hpf struct (n) at 3-6 hpf n (%) (n) n (%) Ik4 379 361316 183 27 (13%) (87.5%) Ik2wt 312 303 256 235 11 (4.5%) (84.5%) act 1620 146 134  5 (3.7%) control (90.1%)*including embryos taken for analysis

A lack of pigmentation (a/oligochromemia) in the circulating blood cellsas seen through pericardium, was observed at 48-72 hpf in 27 out of 183(13%) Ik-4 injected embryos and fry. This data is contrasted witha/oligochromenia observed in only 4.5% and 3.7% of the Ik-2 injectedembryos and intact control, respectively.

Example 3 In Situ Localization of nIkaros Transgenes

The topographical profile of the human Ikaros transgenes Ik-4 and Ik-2expressed in the ZF embryos at 17-19 hpf was confirmed by whole-mount insitu hybridization using digoxigenin-labeled Ikaros riboprobes. Probeshybridized to the human Ikaros mRNA were immunolocalized with anti-DIGFab fragments and detected by chromogenic reaction with NBT/BCIP. Nofalse positive signals were detected in non-transgenic control ZFembryos (FIG. 2A). In transgenic ZF embryos, the chromogenic(blue-purple) signal of human Ik-4 or Ik-2 transgene expression waslargely localized to the trunk region containing the intermediate cellmass (ICM) where primitive hematopoiesis takes place (FIGS. 2B-2F),reminiscent of the expression profile of other regulators ofhematopoiesis such as GATA-1 and c-MYB (Detrich et al., 1995, Amatrudaand Zon, 1999).

At 48 hpf, embryonic hematopoiesis in the ZF shifts from the ICM to thedorsal mesentery and forms the “dorsal” fetal hematopoietic site(Detrich et al., 1995, Amatruda and Zon, 1999). At this transition stagefrom primitive to definitive hematopoiesis, the trunk axial vesselcomplex, i.e., dorsal aorta and axial vein, stretches along theanterior-posterior axis, between the notochord and trunk endoderm, andcontains circulating embryonic blood cells (FIG. 3A). ZF embryos derivedfrom the Ik-4 injected and from the Ik-2 injected eggs were fixed withparaformaldehyde at 48 hpf and frozen sagittal section were stained withhematoxylin/eosin or immunostained with the antibodies raised againsthuman Ikaros. Sections were examined by laser confocal microscopy. Themedian saggital section of the trunk region (A) of the 48 hpf ZF embryowith anterior to the left and dorsal to the top of the frame, provides alayout of the “dorsal” hematopoietic site with dorsal aorta (DA) andaxial vein (AV) with mesenchyme hematopoietic cells of the dorsal aortaventral wall (M) and circulating primitive blood cells (PB). From thedorsal side the axial major vessel complex are bordered by the notochord(NC) and neural tube (NT); from the ventral side the vessels areneighbored by the endoderm (E) and the yolk extension (YE).

Examination of the sagittal sections of the trunk region in Ikarostransgenic ZF embryos by immunofluorescence staining with antibodiesdirected against human Ikaros and confocal laser scanning microscopyshowed expression of human Ikaros in circulating lymphohematopoieticcells (FIG. 3B) as well as in the cells of the ventral wall of dorsalaorta (FIG. 3C) and ventral vein region (data not shown). Human Ik-4isoform showed cytoplasmic and patchy nuclear expression inhematopoietic cells of transgenic ZF embryos (FIGS. 3B-3C), reminiscentof its intracellulal localization pattern in human cells (Sun et al.,1999). A similar pattern of subcellular compartmentalization wasobserved in circulating ZF blood cells expressing the Ik-2 isoform. Ik-2protein was detected in the nuclei and cytoplasm of the blood cells(FIG. 3D). No false positive signals were detected in non-transgeniccontrol ZF embryos (FIG. 3E).

Example 4 Deregulated Expression of GATA-1, c-MYB and lck Genes in HumanIk-4 and Ik-2 Transgenic ZF Embryos

The impact of Ik-4 and Ik-2 transgene expression in the ZF embryos onthe expression pattern of the early hematopoietic and lymphopoieticmarkers GATA-1, c-MYB, Rag-1, and ick was evaluated. Ik-4 and Ik-2transgene expression interferes with the normal expression of ZF GATA-1,c-MYB, and ick, but not with Rag-1. The zinc-finger transcription factorGATA-1 is one of the central regulators in hematopoietic celldifferentiation within the myeloid and erythroid lineages (as thoroughlyreviewed by Orkin and Zon, 1997). The transcriptional regulator ofmyelopoiesis encoded by the proto-oncogene c-MYB and its target c-mychave been implicated in myeloid leukemogenesis, as reviewed by Wolff,1996 and Weston, 1999. Both GATA-1 and c-MYB are expressed in the ZFduring 15-24 hpf and they strongly demarcate the forming ICM, theearliest site of primitive hematopoiesis (Detrich et al., 1995, Liao etal., 1998, Bahary and Zon, 1998).

By comparison, expression of the lymphoid marker Rag-1 in the ZFcommences at 3 dpf, when thymocyte precursors seed the bi-lateral thymicanlage (Trede and Zon, 1998). A similar pattern of expression restrictedto bilateral thymi was shown for lck (Dr. Nikolaus Trede, personalcommunication).

Expression patterns of the ZF early hematopoietic genes in the presenceof Ik-4 and Ik-2 transgene expression were studied in the ZF embryos bymeans of two color/fluorescence whole-mount in situ hybridization. Allimages are positioned anterior to the top and dorsal side to the rightof the frame.

In contrast to the first two markers, Rag-1 expression is restricted tothymocytes after they seed the thymus anlagae at 3 dpf. We observedRag-1 expression in the bi-lateral thymi at 4 dpf in all tested fishfrom the Ik-4 injected and Ik-2 injected groups and in the control withno regard to the transgene expression. Rag-1 was transcribedbi-laterally in the location of thymus primordial.

Following two color/fluorescence in situ hybridization with human Ikarosand ZF GATA-1 riboprobes, all non-injected control embryos at 17 hpfwere found positive for GATA-1 expression and showed no false positivesignals of human Ikaros expression (FIG. 4A). GATA-1 positive cellsformed a distinct strip of the ICM in the trunk region between thesomite mesoderm and the yolk protrusion, which was remarkably vivid withthe use of fluorescence (FIG. 4D). Notably, in the ZF embryos expressingthe human Ik-4 transgene, the GATA-1 positive ICM region was much largerthan in non-injected control embryos (FIGS. 4B & 4E). In contrast, inthe ZF embryos expressing the Ik-2 transgene, the GATA-1 positive cellsin the ICM site were few and formed a dotted rather than a solid line(FIGS. 4C & 4F).

In all control embryos tested with human Ikaros and ZF c-MYB riboprobesat 15 hpf, c-MYB-positive cells were condensed in the distinct region ofthe ICM (FIG. 4G). Similar to GATA-1, the region of c-MYB positive cellswas visibly enlarged in the Ik-4 transgenic ZF embryos (FIG. 4H), anddramatically decreased in the Ik-2 transgenic embryos with expressionrestricted to the uttermost caudal portion of the ICM (FIG. 41).

In contrast, expression of Rag-1 was not altered in the Ik-4 nor in theIk-2 transgenic embryos. In all embryos, positive fluorescent signalswhich mark Rag-1 expression were restricted at 4 dpf to the bi-lateralsites of developing thymus, and were localized to the region ofpharyngeal arches between the eye and pectoral fin (FIGS. 4J, 4J, 4L).Similar to Rag-1, the ZF lck expression in the control non-injected ZFlarvae at 4-5 dpf was restricted to the thymic location (FIG. 4M). Thelck expression was not affected in the Ik-4 injected larvae (FIG. 4N)but was dramatically decreased or totally absent in the Ik-2 injected ZFlarvae at 4-5 dpf (FIG. 4O).

Example 5 Abnormal Hematopoiesis with Lymphoid Hyperplasia in Adult ZFDerived from Ikaros 4 Transgenic Embryos

Adult fish derived from the Ik-4 and Ik-2 transgenic embryos as well asfrom non-injected control embryos were raised in similar conditions as 3separate groups. At 3 months of age, these fish were all in apparentgood health with normal shape and coloration, and reached maturityaccording to the breeding behavior and pair-wise mating. The bodyweight, body length, liver size and spleen size of Ik-4 and Ik-2injected ZF were not different from those of the adult non-transgeniccontrol fish (Table 2). TABLE 2 Analysis of Adult Fish n Mean(±SEM)Median Range p-value Difference Length Ik-4 18 3.74 ± 0.07 3.8 3.15-4.100.48 NO Ik-2 22 3.53 ± 0.05 3.5 3.0-4.0 0.18 NO intact control 8 3.76 ±0.22 3.95 2.75-4.5  Weight Ik-4 18 491.71 ± 32.4  531.25 274.92-704.290.28 NO Ik-2 22 416.87 ± 30.32  367.49 272.28-862.7  0.28 NO intactcontrol 8 454.19 ± 52.59  482.52 181.68-651.39 Liver/total wt Ik-4 182.36 ± 0.32 1.57   1-4.98 0.29 NO Ik-2 22 2.06 ± 0.32 1.57 0.65-6.560.43 NO intact control 8 2.13 ± 0.27 2.25 0.89-3.1  Spleen/total wt Ik-417 0.08 ± 0.01 0.08 0.03-0.17 0.35 NO Ik-2 19 0.11 ± 0.01 0.11 0.03-0.210.15 NO intact control 6 0.09 ± 0.02 0.1 0.04-0.13

In adult ZF, kidney plays the role of the bone marrow in mammals. Kidneyhematopoietic cells from 50 mature 3-month-old adult ZF derived from theIk-4 transgenic, Ik-2 transgenic, and intact embryos were imprinted ontoslides and differentially stained with Wright/Giemsa for microscopicexamination of cellularity and cellular composition. Hematopoietic cellindexes were calculated for 18 adult ZF derived from the Ik-4 injectedembryos, 22 adult ZF derived from the Ik-2 injected embryos, and 10adult ZF derived from the control non-injected embryos.

To examine representation of hematopoietic cell types in adult fish,kidneys extracted from 18 Ik-4 injected, 22 Ik-2 injected and 10non-injected adult ZF were imprinted on slides and staineddifferentially with Wright/Giemsa. All samples contained a multilineagepopulation of hematopoietic cells including both progenitors and matureforms (Table 2, FIG. 5A).

Imprints from the control, non-injected ZF showed multilineagehematopoiesis with marked myeloid (43%) and erythroid (30%) hyperplasia.Other cell types identified in the imprints consisted of lymphoid cells(15%), granulocytes (10%) and monocytes (5%) (Table 2, FIGS. 5A-5B). Incontrast, in the ZF derived from the Ik-4 injected embryos, lymphoidcells represented 49% of hematopoietic cell population, whereas myeloidand erythroid cells as well as granulocytes were reduced in numbers(24%, 21%, and 3%, respectively), and the number of monocytes remainedunchanged (3%) (Table 2, FIGS. 5A & 5D). In the ZF derived from the Ik-2injected embryos, the number of erythroid cells was drasticallyincreased (46%), while cells of the myeloid and lymphoid lineages werepresent in reduced numbers (29% and 8%, respectively). The number ofgranulocytes was slightly increased (13%), and the number of monocyteswas not markedly changed (4%) (Table 2, FIGS. 5A & 5C).

The data described herein provides evidence that the human leukemogenicdominant-negative isoform Ik-4 and human DNA-binding isoform Ik-2 aretransiently expressed in ZF embryos during primitive and “fetal”definitive hematopoiesis. Early ZF hematopoietic transcription factorsGATA-1 and c-MYB are affected by Ik-4 and Ik-2 expression in oppositeways; they are upregulated by Ik-4 and downregulated by Ik-2 expression.

In adult kidney, distinct lymphoproliferative disorder was observed inZF derived from the Ik-4 transgenic embryos, and erythroproliferativedisorder was detected in ZF derived from the Ik-2 transgenic embryos.The observed phenomenon can be described as a hyperplasia of specificblood cell types which occurs at the expense of other cell lineages as alate response to the Ik-4 and Ik-2 transgene expression during embryonichematopoiesis. Accordingly, the data supports the transgenic ZF as auseful experimental model to study leukemogenesis, Lymphoproliferativedisorders (e.g. leukemias) and erythroproliferative disorders (e.g.anemia).

Discussion

All vertebrates including mammals and fish, utilize the same basicprinciples and share the same major steps of blood development, withwaves of primitive and definitive hematopoiesis, successive changes ofhematopoietic sites in ontogeny, and colonization of hemopoietic organsby blood cell precursors of specific lineages. In this respect, the ZFprovides an excellent model to define genes and genetic pathwaysessential for blood cell differentiation and development ofhematopoietic disorders.

In several large-scale chemical mutagenesis screens, over 50 mutationswere identified in the ZF which affect differentiation in red cell(Ransom et al., 1996, Weinstein et al., 1996) and white cell lineages(Trede and Zon, 1998, Dr. Nikolaus Trede, personal communication). Ithas been shown recently that disruption of the sau gene, which leads tomicrocytic hypochromic anemia phenotype in ZF corresponds to impairedgene coding for erythroid-specific d-aminolevulinate synthase(ALAS2/ALAS-E) necessary for heme biosynthesis, and results incongenital sideroblastic anemia in humans (Brownlie et al., 1998).Mutation of another gene, yqe^(tp61), leads in the ZF to aphotosensitive porphyria. This was linked to uroporphyrinogendecarboxylase (UROD)-deficiency which causes hepatoerythropieticporphyria in humans (Wang et al., 1998). A spontaneous blood mutation,cloche (clo), was found to affect both blood and endothelial celldifferentiation in ZF (Stainier et al., 1995), most probably bydisrupting normal SCL (Tal-1) expression in hemangioblasts (Liao et al.,1998, Gering et al., 1998). These findings demonstrate that at leastsome ZF blood mutations serve as models for human blood disorders.

In support of the ZF as a model animal, it is noted that feasible andreliable vehicles necessary for either transient transgene expression orstable integration and expression are well developed for the ZF. Inaddition, the data recited herein demonstrates that two isoforms of thehuman Ikaros gene that play a critical role in lymphocytedifferentiation were expressed in the ZF during embryonic hematopoiesis.Transgene expression was regulated in the ZF cells, for it started afterthe onset of the zygotic genome transcription. It persisted for thefirst two days in 100% of the injected embryos and in approximately 50%and 40% of the 3 dpf embryos and 4 dpf larvae, respectively.

The β-actin promoter, cloned into the all-fish expression cassette haveused herein was intended to drive transgene expression in all types offish cells. Expression was detected in a variety of cells includingmesenchyme cells of dorsal aorta ventral wall and ventral vein region aswell as in circulating blood cells. Thus, transgene activity in theappropriate cells with appropriate micro-environment may be the cause offuture hematopoietic alteration.

A layout of blood development in the ZF embryo served as a necessarybackground for the present study. In the ZF, the first cells committedto blood differentiation were defined as early as the end ofgastrulation as two lateral stripes of ventral mesoderm with cells(hemangioblasts) expressing, as shown by in situ hybridization, earlyhematopoietic and vasculogenic markers SCL, GATA-1, GATA-2, c-MYB andLMO2 (Detrich et al., 1995, Gering et al., 1998, Liao et al., 1998,Thompson et al., 1998, Amatruda and Zon, 1999). These cells migrate tosomite mesoderm to form in about 2 hours (the 5 somite stage) theintermediate cell mass (ICM). The ICM is known to be the site ofprimitive hematopoiesis in fish (Al-Adhami and Kunz, 1977, Detrich etal., 1995; Willett et al., 1999) and comprises hematopoietic (primarilyembryonic erythroblasts), vasculogenic cells as well as pronephric cellprecursors (Zon, 1995, Weinstein et al., 1996, Liao et al., 1998,Thompson et al., 1998, Willett et al., 1999). The ICM declines with theproduction of circulating erythroblasts and erythrocytes and by 30 hpfhematopoiesis shifts to the nascent “dorsal” site possibly the firstsites of definitive hematopoiesis, the dorsal aorta and to the “ventralvein region” containing blood cell precursors in the axial vein wallsand surrounding mesenchyme (Liao et al., 1998, Thompson et al., 1998,Willett et al., 1999). From here two separate seedings take place: ofthymus (at 65 hpf) and of kidney primordia (starting at 96 hpf) (Hansenand Zapata, 1998, Trede and Zon, 1998, Willett et al., 1999). Whilethymus is colonized by T-lymphocyte precursors, pronephros is seededwith different lineage progenitors including erythro-, myelo- andB-lymphocytes. Finally, with kidney differentiation into head kidney(pronephros) and trunk kidney (mesonephros), the main multilineagehematopoietic site in adults is formed which is unequivocally consideredto be a bone marrow equivalent (Rowley et al., 1988, Hansen and Zapata,1998).

Interaction of the Human Ikaros Isoforms with the ZF HematopoieticGenes.

The data presented herein shows that the area of ZF GATA-1 expression inthe Ik-4-positive embryos at 17 hpf was markedly increased than in thecontrol and Ik-4 negative embryos. Taking into consideration that GATA-1is the earliest marker to be expressed in blood cell progenitors, thisshift in GATA-1 expression pattern suggests enlargement of the whole ICMregion in the embryo. The effect of the Ik-2 transgene was totallyopposite—the GATA-1 positive ICM area was drastically reduced.Similarly, human Ikaros isoforms affected expression of the ZF c-MYB. Inthe Ik-4 transgenic embryos at 15 hpf, c-MYB-positive area in the ICMwas enlarged whereas in the Ik-2 transgenic embryos, the strip ofc-MYB-positive cells was mostly missing showing a decline in c-MYBexpression. Finally, in Ik-2 injected ZF larvae at 4 and 5 dpf, Ickexpression in thymocytes was visibly reduced or totally blocked. Itshould be noted tha in ZF embryos, expression of ZF Ikaros gene wasdetected by in situ hybridization in the ICM (at 5 somite stage and at24 hpf) and then in the dorsal aorta at 46 hpf (Kawasaki et al., 1998).Thus, in addition to a role in determination of hematopoietic stem cellcommitment to lymphoid lineage in adults (Hansen et al., 1997), Ikarosmarks in the ZF embryo, the earliest hematopoietic lymphoid progenitors.

In humans and fish (trout), Ikaros was found to be highly conservedshowing 75% homology in amino acid sequence and 92-98% identity in theactive sites of the protein (Hansen et al., 1997). Structural similaritygives grounds to assume that both human Ikaros isoforms can interactwith endogenous ZF Ikaros, as well as with other ZF genes involved inblood cell differentiation. Overexpression of DNA-binding Ikaros isoformin the blood cell progenitors as well as the occurrence of thenon-binding isoform equally affects blood development.

Distribution of Transgene Expression.

Ik-4 and Ik-2 transgenes were expressed in the ZF embryos in a mosaicfashion. As evidenced by whole-mount in situ hybridization, human IkarosRNA resided in various regions of the embryo but most commonly in thetrunk area, in close proximity to the ICM site. Transgene expression wasconfirmed by immunostaining and human Ikaros isoforms were localized inthe 48 embryos to a number of hematopoietic and nonhematopoietic cells.Along with the sites of ectopic expression, the trunk region of theembryo which at this stage contains the dorsal aorta and axial veincomplex was commonly found positive also.

Human Ik-4 isoform was detected in the circulating blood cells, as wellas in the hematopoietic cells of dorsal aorta ventral wall and cellssurrounding the caudal portion of the axial vein, known as a ventralhematopoietic site (Liao et al., 1998). Human Ik-2 isoform was detectedin circulating blood cells as well as in endothelial cells of the axialvessels. Large hematopoietic cells of the “dorsal” site as well assimilar large cells in the blood stream retained Ik-4 protein in thenucleus and in the cytoplasm, whereas the smaller and much more roundcells of primitive blood retained no signal in the nucleus supposedlydue to its inactivation. Ik-2 protein was localized to the nucleus andto the cytoplasm of the circulating blood cells.

The data show that human Ikaros gene expression in the transgenic ZFembryo may be both ectopic and site-specific. While in non-hematopoieticcells Ik-4 and Ik-2 transgene activity is probably irrelevant to blooddevelopment, their action in the ICM and the “dorsal” sites of embryonichematopoiesis may cause significant changes in the pattern of endogenousZF Ikaros expression with dramatic consequences for blood celldifferentiation. The observed changes in the GATA-1, c-MYB, and Ickexpression patterns suggest that directly or indirectly the activity ofthese early blood cell markers was affected by the transgenes.

To avoid ectopic expression, transgenes may be targeted to specific celltypes. Recent study of the ZF GATA-1 promoter showed that positive andnegative cis-regulatory elements are essential for erythroid-specificexpression (Meng et al., 1999). Promoters from lymphoid-specific ZFgenes (Rag 1,2, lck, Ikaros) can be used to force transgene expressionexclusively in one of the cell lineages. By choosing a cell-specificgene promoter, transgene activation can be restricted to desired celltypes.

Primitive Blood Circulation.

It was generally accepted that embryonic erythrocytes form the onlypopulation of circulating blood cells in the 24-48 hpf ZF embryos.However, large non-erythroid cells defined as granulocytes according totheir ultrastructure, were found in the blood stream of the 48 hpf ZFembryos (Lieschke et al., 1999). This finding make it possible to assumethat other types of blood cells, including lymphoid progenitors may bepresent in circulating blood in the ZF embryo. The data presented hereinshows that in the 48 hpf transgenic ZF embryos, both Ik-4 and Ik-2 humanIkaros isoforms reside in the nuclei and cytoplasm of largenon-differentiated cells found in circulation, as well as in thenon-differentiated cells of the dorsal aorta ventral wall and ventralvein mesenchyme cells. These cells, affected by either dominant-negativeIk 4 expression or by overexpression of the DNA-binding Ik-2 isoform,may comprise the cell pool which seeds the kidney primordium, and thus,be responsible for future changes in adult hematopoiesis.

Blood Cell Types in Adult Fish.

Hematopoietic tissue in the ZF kidney is formed by cords of cells whichsurround blood vessels, in-between the renal tubules and glomeruli(Willett et al., 1999). Erythrocyte, granulocyte, lymphocyte, andmonocyte differentiation has been reported in the adult fish kidney(Rowley et al., 1988) and with the exception of nucleated erythrocytes,the morphology of the other mature and differentiating hematopoieticcells closely resembles that of their mammalian counterparts.Morphological description of fish blood cells, including theirultrastructure and functions is summarized in Rowley et al., 1988.

There are two subpopulations of lymphocytes in fish with differentimmunological properties as reviewed by Miller et al., 1998, whichprecisely correspond to T- and B-cells. T-lymphocytes are locatedpredominantly in thymus where Ikaros (Hansen and Zapata, 1998), Rag 1,2,and lck, a src-family protein tyrosine kinase implicated in T-cellmaturation and activation (Trede and Zon, 1998) are expressed.B-lymphocytes are generated in the kidney; in trout and ZF, Ikaros(Hansen et al., 1997), Rag-1 and Rag-2 (Willett et al., 1997) and TdT(Hansen, 1997) expression in the pronephros was used to confirm thepresence of pre-B-cells. A tec-family non-receptor tyrosine kinaseexpression was found recently in the ZF kidney (Haire et al., 1998)which may represent the Btk expression in the B-lymphocytes.

Neoplasia in Fish.

For several reasons, neoplastic transformation in fish is not widelyreported in the literature. Nonetheless, fish are susceptible toneoplasms and as models were successfully utilized in the studies ofcarcinogenic and teratogenic effects of aquatic pollutants (Pliss etal., 1982, Mizell and Romig, 1997, Oberemm, 2000). In this respect, fishare especially noted for experimentally-induced neoplastic responses inliver reviewed by Hinton and Couch, 1998). Besides hepatocarcinomas,such as in tilapia (Ding et al., 1989), there were reported cases ofolfactory neuroepithelioma in domestic carp (Ishikawa et al., 1978) andabdominal sarcoma in koi carp (Lewbart et al., 1998), plasmacytoidleukemia of a retroviral origin in chinook salmon (Kent et al., 1997)and lymphosarcoma of unknown origin in brook trout (Earnest-Koons etal., 1997). Quite separately stands a very elaborate study of malignantmelanomas in platyfish caused by a dominant oncogene ONC-Xmark which isa thyrosine kinase receptor gene (Schartl et al., 1985). A model formelanogenesis and tumor formation in fish in particular, was proposed(Morizot et al., 1998). Accordingly, the literature supports the studycancerogenesis in small fish model systems, such as ZF. Recently, ZF wasproposed as a model for human blood disorders such as congenitalsideroblastic anemia and hepatoerythropoietic porphyria (Brownlie etal., 1998), (Wang et al., 1998). The data presented herein indicatesthat the ZF may serve as an experimental model of leukemia developmentas well.

FIG. 3A-3B demonstrate this pheonotypic change. FIG. 3A shows a normallypigmented larva, while FIG. 3B shows a larva with a/oligochromemia.Primitive circulating erythrocytes extracted from the fry witha/oligichromemia were indistinguishable in size and shape from thenormal ones, and the loss of pigmentation was the only alteration ofblood cell phenotype observed in these fish. Fry with oligochromemiawere found viable and were raised and further analyzed separately fromthe others.

Example 6 Screening Potential Candidate Drugs for TherapeuticallyEffective Agent

The animal model of the invention can be used to screen candidatecompounds for therapeutic utility in the treatment and/or prevention oflymphatopoietic and hemapoietic disorders, including leukemias. Asdiscussed in the examples above, the insertion of the transgene IK4 intozebrafish embryos markedly altered the normal cellular differentationpattern and is correleated with the development of hematopoieticdisorders, including multilineage hematopoiesis, erythroid hyperplasia,and the like.

Administration of a putative therapeutic agent to the animal modelprovides an efficient, cost-effective, and reliable method for screeingagents for candidates likely to improve outcome and symptoms ofhematopoietic disorders, including leukemias.

Example 7 Screening Potential Carcinogens

The animal model for the invention can be used to screen suspectedcarcinogenic agents, or agents suspected of inducing lymphatopoietic orhepatapoietic disorders. For example, ZF embryos expressing the Ik-2transgenes produce and develop normal B- and T-lineage cells. Agentsthat might disrupt normal Ik-2 or other regulating controls for normalhematopoietic cell development can be efficiently and rapidly screenedby administering the suspected agent to the embryo, as demonstrated byadministration of Ik-4 in the Examples above.

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1. An animal model for lymphocyte development and leukemia, comprising atransgenic zebrafish expressing a heterologous Ikaros protein.
 2. Theanimal model of claim 1, wherein the Ikaros protein is a non-DNA bindingform of Ikaros.
 3. The animal model of claim 2, wherein the Ikarosprotein lacks at least one N-terminal zinc finger domain as comparedwith DNA-binding forms of Ikaros.
 4. The animal model of claim 3,wherein the Ikaros protein is one or more of Ik-4, Ik-5, Ik-6, Ik-7, andIk-8.
 5. The animal model of claim 3, wherein the Ikaros protein is adeletion mutant lacking the following Ikaros amino acid sequence:KSSMPQKFLG [SEQ ID NO: 5].
 6. The animal model of claim 3, wherein theIkaros protein contains an insertion of the following amino acidsequence: VTVGADDFRDFHAIIPKSFSR [SEQ ID NO: 6].
 7. An assay method forscreening potential therapeutic agents useful for treating or preventinghematopoietic disorders, the assay comprising contacting a transgeniczebrafish embryo with a potential therapeutic agent, the transgeniczebrafish embryo expressing a non-DNA binding form of Ikaros, andcorrelating improved lymphohematopoiesis versus a non-treated controlwith an effective therapeutic agent.
 8. The assay of claim 7, whereinthe non-DNA binding form of Ikaros protein lacks at least one N-terminalzinc finger domain as compared with DNA-binding forms of Ikaros.
 9. Theassay of claim 7, wherein the Ikaros protein is one or more of Ik-4,Ik-5, Ik-6, Ik-7, and Ik-8.
 10. The assay of claim 7, wherein the Ikarosprotein is a deletion mutant lacking the following Ikaros amino acidsequence: KSSMPQKFLG [SEQ ID NO: 5]
 11. The assay of claim 7, whereinthe Ikaros protein contains an insertion of the following amino acidsequence: VTVGADDFRDFHAIIPKSFSR [SEQ ID NO: 6].
 12. The assay of claim7, wherein said improved lymphohematopoiesis is analyzed in thedeveloping zebrafish embryo.
 13. The assay of claim 12, wherein saidimproved lymphohematopoiesis comprises improved oligochromemia in thecirculating blood cells of the animal model.
 14. The assay of claim 7,wherein said improved lymphohematopoiesis is analyzed in adultzebrafish.
 15. The assay of claim 7, wherein said improvedlymphohematopoiesis comprises improved cellularity and celllularcomposition of adult zebrafish kidney imprints.
 16. The assay of claim7, wherein said improved lymphohematopoiesis comprises one or more oflessened multilineage hematopoiesis, reduced erythroid hyperplasia, andreduced numbers of lymphoblasts as compared with control animals.